Coffee is prepared from the roasted beans of the plants of the genus Coffea, generally from the species C. arabica (Caturra coffee) and C. canephora (Robusta coffee), and hybrids of these. Beans are the seeds of the coffee plant and are obtained by processing the coffee fruit, ideally the mature coffee fruit which commands the best price due to its superior quality. In order to obtain high quality xe2x80x9cgourmetxe2x80x9d coffee, it was considered necessary in the past to pick the coffee tree fruit by hand because the fruits of a coffee tree do not ripen uniformly and, thus, there are both mature and immature fruit on the same tree. This did not previously present a serious problem, as most coffee is grown in areas of the world where labor is plentiful and not expensive. However, recently, a lack of abundant and inexpensive labor has bercome a major contributor to decreased coffee production. In order to increase productivity, countries in some regions of the world, such as the largest coffee producing country, Brazil, have resorted to strip harvesting where workers rapidly remove all fruit from a branch whether ripe or unripe. The speed of harvesting is thus increased, but the yield of the highest quality beans is decreased because much of the harvested fruit is immature (green).
The lack of uniform ripening of coffee fruit on the tree has also seriously limited the effectiveness of mechanical harvesting. The force required to remove mature fruit (cherry) from the tree is similar to the force required to remove green fruit. Thus, mechanical harvesters do not distinguish well between green fruit and cherry and a large amount of immature fruit is harvested along with mature fruit. If coffee fruit ripening could be controlled so that all fruit ripened at one time, both the strip method of hand harvesting and mechanical harvesting would be much more efficient and a higher percentage of the harvested fruit would be in the higher quality grades, resulting in increased profitability of coffee production.
Ripening of fruit involves a number of changes in the fruit. In fleshy fruits, chlorophyll is degraded and other pigments often form, changing the color of the fruit. Simultaneously, the fleshy part softens as a result of the enzymatic digestion of pectin, the principal component of the middle lamella of the cell wall, and starches and organic acids are metabolized into sugars. Fruits are divided into two major groups, based on the respiratory behavior observed during the ripening process. In the climacteric fruits, such as tomatoes, avocados, bananas, apples and pears (i.e., pome fruits), and papaya, there is a large increase in respiration (i.e., a large increase in oxygen uptake termed the xe2x80x9cclimacteric risexe2x80x9d) concomitant with a burst of ethylene synthesis, producing marked changes in fruit composition and texture. In these fruits, the xe2x80x9cclimactericxe2x80x9d is required for the final stages of ripening when softening and development of color and flavor occurs. Other plants do not have a climacteric and ethylene does not seem to be important in their fruit ripening. Such fruits that show a steady decline or gradual ripening are called xe2x80x9cnon-climacteric fruitsxe2x80x9d (e.g., citrus, grapes, watermelon, cherries, pineapples, strawberries, and most vegetable crops such as carrots, onions, celery, spinach, crucifers, peas and beans).
Once climacteric fruit reach a certain stage of maturity, it is known that they can be induced to ripen by the exogenous application of ethylene, such as during storage and/or transport. Techniques to avoid exposure of climacteric fruits to ethylene until just before marketing have been used to control and regulate the timing of the ripening process, and have had a major impact on the quality of fruit sold. For example, tomatoes are often picked when they are green, and then stored in the absence of ethylene until just before marketing, at which time they are exposed to exogenous ethylene to induce simultaneous ripening. Exogenous ethylene has also been used commercially to promote loosening of fruit such as cherries, blackberries, grapes, and blueberries, thereby facilitating mechanical harvesting of these fruit crops.
In view of the foregoing, it would be very advantageous to be able to characterize coffee plants as to whether or not they are climacteric and, if shown to be climacteric, to control the ripening of coffee fruit by exogenously applied ethylene. Until the investigations described herein and in our co-owned U.S. Pat. No. 5,874,269, the disclosure of which is hereby incorporated by reference in its entirety, it was not known whether coffee fruit is climacteric or non-climacteric. Although it was observed that coffee fruit ripened in response to ethylene after reaching a certain stage of development [Crisosto, C. H., et al., J. Haw. Pac. Agri. 3:13-17 (1991)], it was not possible to measure ethylene evolution or a respiration increase in ripening fruit. This may be because of the small size of the fruit and the lack of uniformity of ripening.
The biosynthesis of ethylene begins with the reaction of methionine and ATP to form S-adenosylmethionine (SAM). The enzyme ACC synthase catalyzes the conversion of SAM to 1-aminocyclopropane-1-carboxylic acid (ACC). In most plants this is the rate limiting step. The ACC is then converted to ethylene, in a reaction that is catalyzed by ACC oxidase [Yang and Hoffman, Ann. Rev. Physiol. 35, 155 (1984)].
It is well known that ethylene is related to various events in plant growth and development, including fruit ripening, seed germination, leaf and flower senescence and abscission, and root and leaf growth. Ethylene production is strictly regulated by the plant and can be induced by a variety of external stress factors, including the application of auxins, wounding, anaerobic conditions, viral infection, chilling, drought, ions such as cadmium and lithium ions, and the like.
Recombinant DNA technology has been used to isolate a number of ACC synthase genes from, for example, rice, petunia, winter squash, zucchini, tomato, tobacco, mung bean, soybean, and apple. Examples of these ACC synthase genes are described in our co-owned U.S. Pat. No. 5,767,376, the disclosure of which related to these examples is hereby incorporated by reference. However, with the exception of the apple and a subset of the tomato ACC synthase gene sequences, none of the described ACC synthase genes are involved with the ripening of fruit. Therefore, ethylene production in plants is apparently governed by a family of ACC synthase genes, at least in the above examples, not all of which are expressed during fruit ripening, e.g., some would be active in wound response, and the like. Similarly, it is considered likely that there is a family of ACC oxidase genes in plants that are variously active at different stages of plant growth and fruit ripening. The DNA sequences of the members of the ACC synthase gene family or the members of the ACC oxidase gene family in a plant such as coffee are therefore thought to be different from each other, although they would be related. For example, ACC synthase is encoded by at least six divergent genes in tomato. J. E. Lincoln et al. [J. Biol. Chem. 268 (no. 26), pp. 19422+, September 1993] compared the gene sequences of two ACC synthases thought to be involved in fruit ripening in tomatoes and found a sequence homology of only 71%. Two other ACC synthase genes from tomato had a sequence homology of 96% with each other. However, the sequence homology between the two sets of ACC synthase genes was only 68% and they had only a 51% sequence homology with an ACC synthase gene from rice. It is similarly expected that the genes coding for ACC synthases involved in fruit ripening from different varieties of coffee, such as C. arabica, C. canephora, and blends of these, such as the Timor hybrid and the like, would show a high sequence homology, but would not be identical. Moreover, the findings in the tomato demonstrate the importance of using ripening coffee fruit tissue in order to be able to isolate genes coding for any ACC synthase(s) that are expressed during fruit ripening, because these genes are likely to be different than ACC synthase genes expressed during other phases of the growth of the coffee tree.
A strategy for determining whether coffee trees are climacteric would be to measure the level of expression of the xe2x80x9cfruit ripeningxe2x80x9d ACC synthase gene and/or the xe2x80x9cfruit ripeningxe2x80x9d ACC oxidase gene, i.e., by measuring the levels of messenger RNA (mRNA) coding for each of these enzymes, during fruit ripening. Once it has been established that coffee plants are climacteric, a further strategy to control the ripening of coffee fruit would be to prevent synthesis of the specific ACC synthase enzyme and/or the ACC oxidase enzyme in the pathway for ethylene biosynthesis during fruit ripening and to apply exogenous ethylene to synchronize and control fruit ripening in coffee plants.
Thus, in one embodiment of the invention, coffee plants are genetically altered to eliminate synthesis of ACC synthase; in another embodiment, ACC oxidase synthesis is eliminated; and in another embodiment, synthesis of both enzymes is eliminated. In the presently preferred embodiments, synthesis of one or both of these enzymes is eliminated by transforming coffee plants with a nucleic acid sequence that codes on transcription for an RNA that is antisense to the mRNA that codes on expression for the enzyme whose synthesis is to be eliminated. See Oeller et al., Science 254:437 (1991), who reported controlling ripening of tomatoes using a similar strategy. In another embodiment of the invention, synthesis of one or both of ACC synthase and ACC oxidase is eliminated by transforming coffee plants with a nucleic acid sequence that codes on transcription for an RNA that is sense to the mRNA that codes on expression for the enzyme whose synthesis is to be eliminated. Such a strategy is well known and is termed co-suppression or sense-suppression.
Although recombinant DNA technology has been used to isolate a number of ACC synthase and ACC oxidase genes from other plants, it was not until the present invention that genes for ACC synthase and ACC oxidase enzymes that are active in coffee fruit ripening have been identified, isolated and sequenced. An important reason why these genes have not been previously identified, isolated and sequenced is that coffee fruit contains high levels of phenolic compounds such as chlorogenic acid (5-O-caffeoylquinic acid), and high levels of carbohydrates. For example, depending on the coffee variety, ripe seeds of Coffea arabica L. contain between 4% and 8% dry weight of chlorogenic acid. [Aerts, R. J. and T. W. Baumann. J. Exp. Botany 45, 497-503 (1994)]. In another study of 12 different cultivars of Coffea arabica L, the average content of phenolic compounds tentatively identified by HPLC in fresh coffee pulp was 42.2% chlorogenic acid, 21.6% epicatechin, 5.7% isochlorogenic acid I, 19.3% isochlorogenic acid II, 4.4% isochlorogenic acid III, 2.2% catechin, 2.1% rutin, 1.6% protocatechuic acid, and 1.0% ferulic acid. When the percentages of chlorogenic and isochlorogenic acids were added to the corresponding one of epicatechin for each cultivar, it was found that these acids made up between 92.0% and 98.4% of the total of identified phenolic compounds. [Ramirez-Martinez, J. R., J. Sci. Food and Agriculture 43, 135-144 (1988)]. In another study reported at the 13th International scientific colloquium on coffee in 1989, Ramirez-Martinez reported the nature and content of phenolic acids extracted with hot 70% methanol from sun-dried pulp of Robusta coffee (Coffea canephora), Red Bourbon, Caturra (Coffea arabica), Timor hybrid (C. canephoraxc3x97C. arabica) and Catimor (Timor hybridxc3x97Red Caturra) berries (ripe coffee seeds), the total chlorogenic acid content was 4 times higher in Robusta (1.6%) than in Timor hybrid and Catimor, whereas the C. arabica cultivars had intermediate values. Therefore, it appears that the coffee fruit from most, if not all, commercially important species of coffee contain high levels of phenolic compounds.
The combination in the coffee fruit of high levels of phenolics and high levels of carbohydrates makes it very difficult to obtain clean preparations of RNA from this tissue. For example, homogenization of tissue in homogenization buffers typically used to obtain RNA from non-coffee fruit tissues cannot be used for obtaining RNA from coffee fruit because the darkly colored polyphenols in the coffee fruit adhere to the nucleic acids in the tissue during grinding, with the result that the tissue turns dark brown to black. The adhered polyphenol compounds prevent, for example, cutting of the nucleic acids with restriction enzymes, copying of the mRNA with reverse transcriptase to produce a cDNA library, and the like. Moreover, the high levels of carbohydrates prevent the use of a typical chloroform/alcohol-precipitation of RNA from the tissue homogenate because the carbohydrates are co-precipitated with the RNA to produce a carbohydrate-RNA xe2x80x9cglobxe2x80x9d.
Thus, in order to overcome the problems inherent in obtaining clean RNA preparations from coffee fruit, it is necessary to develop new methods for extraction of the RNA that address the high levels of phenolics and carbohydrates in coffee fruit tissue.
The present invention establishes that coffee fruits are climacteric and, therefore, like other climacteric fruits, it is possible to regulate coffee fruit ripening by the application of exogenous ethylene. By developing techniques to isolate substantially pure RNA from coffee fruit, it has been demonstrated herein that mRNA coding for coffee fruit-expressed ACC synthase is present in a small amount in young fruit, and that the level of this ACC synthase mRNA increases rapidly as coffee fruit matures. It has also been demonstrated that accumulation of coffee fruit-expressed ACC oxidase mRNA is similar to that of the ACC synthase mRNA, except that the levels of expression are much higher. The rapid rise in the amount of both ACC synthase and ACC oxidase mRNA during the final stages of fruit ripening in coffee is indicative of a climacteric fruit. It is believed that this invention provides the first convincing evidence that coffee is a climacteric fruit.
Because of the improved methodology described herein for isolating substantially pure mRNA from coffee fruit, cDNA libraries have been constructed from which substantially pure nucleic acid sequences that code on expression for coffee fruit-expressed ACC synthase and ACC oxidase are isolated and sequenced. The invention further provides substantially pure coffee fruit-expressed ACC synthase and ACC oxidase. The invention further provides recombinant nucleic acid sequences, including hosts transformed with such sequences, for transforming coffee plants to suppress the expression of enzymes necessary for ethylene synthesis during coffee fruit ripening. The nucleic acid sequences and recombinant DNA molecules are characterized in that they code on expression for the enzymes ACC synthase or ACC oxidase that are elements of the pathway for ethylene biosynthesis in coffee fruit ripening.
In one embodiment of the invention, coffee plants are transformed with DNA constructs that comprise a transcriptional initiation (promoter) region operably linked to a nucleic acid sequence that codes on a transcription for an RNA that is complementary (antisense) to a substantial run of bases of a mRNA that codes for a coffee fruit-expressed ACC synthase and/or ACC oxidase. In another embodiment of the invention, coffee plants are transformed with DNA constructs that comprise a transcription promoter operably linked to a nucleic acid sequence that codes on transcription for an RNA that shows substantial homology (sense) to a substantial run of bases of the mRNA that codes for coffee fruit-expressed ACC synthase and/or ACC oxidase. Expression of sense or antisense nucleic acid sequences in the transformed plants eliminates the synthesis of ethylene during coffee fruit ripening, although other aspects of cellular metabolism are not affected.
Ripening of the fruit of the transformed plants can be regulated by exogenous ethylene. By application of ethylene to the entire plant, the entire plant will ripen at once, making manual and mechanical harvesting of coffee more productive.